PROJECT SUMMARY Genomic instabilities drive the progression of cancer, aging, and other human diseases. The integrity of our genome is especially at risk while it is being replicated, as replication forks often encounter obstacles to their progression, including DNA lesions, hard-to-replicate sequences, transcription intermediates, or protein-DNA complexes. These encounters often result in DNA breaks, gross chromosomal rearrangements and aneuploidy, which are key events in cancer initiation. The proper repair of stalled or collapsed replication forks through homologous recombination (HR)-based mechanisms plays a major role in preventing replication stress-induced genomic instabilities and many mutations in components of the HR machinery have been associated with cancer predisposition. In the absence of an intact HR-machinery, error-prone mechanisms such as non-homologous end joining (NHEJ) tend to become hyper-utilized, leading to extensive genomic instability. The regulatory basis of the recruitment of HR and NHEJ factors to DNA lesions is therefore of central interest to genome biology and cancer research, not only for explaining the mechanisms of tumorigenesis, but also for providing promising avenues for cancer therapy, as recently demonstrated for PARP inhibitors that are now approved for treatment of ovarian cancer patients with BRCA1 mutations in Europe and the US. This proposal will investigate a new mechanism for regulation of recombinational DNA repair and repair pathway choice. The central hypothesis is that the evolutionarily conserved scaffolding protein TopBP1 plays a central, yet largely unexplored, role in the control of HR-mediated repair and DNA repair pathway choice. Utilizing biochemical, proteomic and genetic approaches in yeast, human cell lines and genome-edited mice, we will define the molecular mechanism by which TopBP1 and its yeast ortholog Dpb11 control DNA repair. Our studies will provide unparalleled molecular understanding of how the action of key HR and NHEJ factors are coordinated and will reveal how signaling networks integrate the control of DNA replication, checkpoint signaling and DNA repair. Proposed experiments will reveal novel mechanisms of repair pathway choice and recombinational repair that are crucial to suppress genomic instability and cancer. Generated outcomes will have implications in the study of tumorigenesis caused by dysfunctions in HR repair and should provide new rationale for therapy.